CN104628864B - Anti-tumor fusion protein EL-defensin, and coding gene and application thereof - Google Patents

Abstract

The invention provides an anti-tumor fusion protein, which has the following primary amino acid sequence structure: EGFR specific targeting oligopeptide-connecting peptide 1-prosthetic group protein LDP-connecting peptide 2-defensin HBD-1; wherein, the EGFR specific targeting oligopeptide has an amino acid sequence shown in SEQ ID NO. 1, the prosthetic group protein LDP has an amino acid sequence shown in SEQ ID NO. 2, and the defensin HBD-1 has an amino acid sequence shown in SEQ ID NO. 3. The fusion protein is a novel anti-tumor fusion protein taking prosthetic group protein LDP as a bracket, and has the functions of EGFR targeting and killing tumor cells by defensin HBD-1. The invention also provides a coding gene and a pharmaceutical application of the fusion protein.

Description

Anti-tumor fusion protein EL-defensin, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of medicinal protein. Specifically, the invention relates to an anti-tumor fusion protein, a coding gene thereof, a medicament using the fusion protein as an active component and pharmaceutical application thereof.

Background

Human defensins (defenses) are produced by granulocytes and epithelial cells and are genetically classified into α -defensins and β -defensins, which appear mainly to kill pathogenic microorganisms and enveloped viruses in terms of innate immunity and to act mainly as chemical inducers and to activate immune cells in terms of acquired immunity α -defensin (HNP 1-3) is overexpressed in tumor cells while β -defensin (HBD) is rarely expressed in tumor cells and recent studies have shown that HBD-1 can act as a tumor suppressor gene, especially in recent years show that HBD-1 has important associations with tumor cell proliferation, differentiation and metastasis, e.g., induction of overexpression of HBD-1 in prostate cancer cells can inhibit the proliferation of prostate DU145 and PC3, lyse cells and initiate caspase protein mediated apoptosis, HBD-1 stimulates oral squamous cell carcinoma cells, resulting in increased expression of HBD-3 in BHY cells and inhibition of proliferation of small cell carcinoma cells, as well as the proliferation of epithelial cells, e-derived from epithelial cell proliferation of epithelial cells, e-26-3623-t 3b, and the proliferation of epithelial cells, which results in hyperproliferation of HIF 3-t.

The prosthetic protein (LDP) is the protein part of Lidamycin LDM and can be prepared by a genetic engineering method. It has been previously reported that LDP can specifically bind to tumor tissues, and itself has a moderate degree of anti-tumor effect in vivo, and is relatively stable in physical and chemical properties. Therefore, the LDP can be used as a stent carrier for developing novel antitumor drugs by virtue of the unique molecular structure and the antitumor property of the LDP.

The Epidermal Growth Factor Receptor (EGFR) is a member of transmembrane receptor tyrosine kinase family (ErbB), and the ligand of the EGFR comprises EGF, TGF- α, Ampheirulin, HB-EGF and the like, the ligand is combined with the receptor, so as to activate a series of intracellular signal channels and finally promote the proliferation, differentiation and migration of cells.

Based on the above, the invention develops a novel anti-tumor fusion protein taking the prosthetic group protein LDP as a bracket, and the novel anti-tumor fusion protein has the functions of EGFR targeting and killing tumor cells by defensin HBD-1.

Disclosure of Invention

The invention aims to provide a fusion protein which takes LDP as a bracket and has defensin (HBD-1) targeting EGFR, and the fusion protein is a medicinal protein with antitumor effect and smaller molecular weight.

Another object of the present invention is to provide a gene encoding the fusion protein.

Still another object of the present invention is to provide a pharmaceutical composition comprising the above fusion protein as an active ingredient and a pharmaceutical use of the above fusion protein.

The invention realizes the aim through the following technical scheme:

to date, in the studies on the antitumor activity of defensins (defensins) reported in foreign literature, defensins (defensins) have been mainly obtained by eukaryotic expression systems. However, defensins contain three disulfide bonds, are not readily soluble and are expressed in very small amounts, making it difficult to obtain high purity defensin molecules. Probably because defensin molecules are difficult to express, few reports about fusion proteins of defensins exist, no reports about fusion proteins of defensins exist at present, and no reports about fusion of defensins with targeting moieties or molecules exist. The inventor constructs a defensin (HBD-1) fusion protein (named as EL-defensin) recombinant vector with target EGFR specificity by taking LDP as a bracket through a genetic engineering technology, and converts the recombinant vector into engineering bacteria BL21star^TM(DE3) and purifying to obtain a multifunctional medicinal protein with anti-tumor effect. Wherein, in addition to the expressed protein content, regarding EGFR targeting, the inventor selects EGF important structure C ring combined with EGFR receptor, 22 amino acid residues on the EGF important structure C ring are used as guide molecules, and the formed protein with smaller molecules can more easily penetrate the extracellular space of the tissue to reach the deep part of the solid tumor.

Accordingly, in one aspect, the present invention provides an anti-tumor fusion protein having the following primary amino acid sequence structure:

EGFR-specific targeting oligopeptide-linker peptide 1-prosthetic protein LDP-linker peptide 2-defensin (HBD-1);

wherein, the EGFR specific targeting oligopeptide has an amino acid sequence shown in SEQ ID NO. 1, and 22 amino acids in total; the prosthetic group protein LDP has an amino acid sequence shown as SEQ ID NO. 2, and has 110 amino acids in total; the defensin HBD-1 has an amino acid sequence shown in SEQ ID NO. 3, and has 36 amino acids in total.

In the fusion protein, EGFR specific targeting oligopeptide is connected with prosthetic group protein LDPLinker 1 and linker 2 linking the prosthetic protein LDP and defensin HBD1 may be the same or different. Preferably, the linker peptide 1 is (GGGGS)₂The connecting peptide 2 is (GGGGS)₂When selecting the linker, it is considered that the linker is too short, it is rigid, it is not favorable for the extension of the protein molecule, it is easy to cause the mutual conceptions of the protein molecule to be hidden, and it is considered that the prosthetic protein LDP as a scaffold has 110 amino acids in total, has α helices and β folds, and has a complicated structure, and thus, it is selected from (GGGGS)₂。

Preferably, the fusion protein has an amino acid sequence shown as SEQ ID NO. 4; or the fusion protein has an amino acid sequence shown as SEQ ID NO. 5;

further preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4 or SEQ ID NO. 5.

SEQ ID NO. 4 is the amino acid sequence of a specific fusion protein provided by the invention, and the total amino acid sequence is 188. From amino terminus to carboxy terminus, the fusion protein comprises: an EGFR-specific targeting oligopeptide (Ec) of 22 amino acids in total; linker 1, is (GGGGS)₂Flexible peptides, total 10 amino acids; the prosthetic protein LDP, total 110 amino acids; linker 2, is (GGGGS)₂Flexible peptides, total 10 amino acids; defensin HBD-1 (HBD-1), 36 amino acids in total.

SEQ ID NO. 5 is the amino acid sequence of another specific fusion protein provided by the present invention, and the total amino acid sequence is 192 amino acids. Compared with SEQ ID NO:4, the fusion protein has Met and Ala before the EGFR specific targeting oligopeptide and Leu and Glu at the C-terminus of the defensin.

In another aspect, the present invention provides a gene encoding the above fusion protein.

Preferably, the coding gene has a nucleotide sequence shown as SEQ ID NO. 6 or 7;

further preferably, the nucleotide sequence of the coding gene is shown as SEQ ID NO 6 or 7.

On the other hand, the invention also provides an anti-tumor medicament, and the medicament takes the fusion protein provided by the invention as an active ingredient. Preferably, the anti-tumor drug is used for treating or preventing squamous cancer, lung cancer or pancreatic cancer, preferably lung cancer.

In another aspect, the invention also provides the use of the fusion protein in preparing an anti-tumor drug. Preferably, the anti-tumor drug is used for treating or preventing squamous cancer, lung cancer or pancreatic cancer, preferably lung cancer.

In summary, the present invention provides a novel fusion protein with killing and targeting properties by applying genetic engineering methods. The fusion protein is a novel protein which targets EGFR (epidermal growth factor receptor) and contains LDP and defensein. Compared with the prior art, the fusion protein has the following substantial differences and effects:

in the prior art, no report has been made on fusion of defensins with other parts or molecules, in particular targeting parts or molecules, to form fusion proteins. The reason for this may be that the human defensin molecule HBD-1 contains three disulfide bonds and is therefore extremely difficult to express in prokaryotic cells, and is often expressed in the form of inclusion bodies, and it is not easily expressed by fusion with other proteins due to its complex molecular spatial structure.

In the fusion protein provided by the invention, the LDP is used as a support, and the defensin is fused with the ligand oligopeptide of the EGFR, wherein the ligand oligopeptide of the EGFR is used as a targeting molecule, and the defensin HBD-1 is used as a warhead, so that the warhead small molecule medicine targeting the EGFR is constructed. This allows direct expression to obtain defensin-containing protein molecules. Experiments prove that the obtained fusion protein not only keeps the effect of the defensin on killing tumor cells, but also can target tumor tissues more specifically, so that the fusion protein has the advantage of better tumor killing effect, has a good anti-tumor effect in an animal body, and has more obvious technical effect compared with the single application of each part. Moreover, even though some reports of constructing fusion proteins by using LDP as a scaffold exist in the field, the existing fusion proteins are often used for enabling LDP to play a role in assembling warhead molecules AE, and are essentially different from the fusion proteins provided by the invention, and often have the defects of high toxicity, no targeting property and the like.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the construction of the recombinant expression plasmid pET 30-el-defensein.

FIG. 2 shows the detection result of recombinant bacterium expression product EL-defensin fusion protein. Wherein:

FIG. 2A is an electropherogram of EL-defensin localization analysis, 1-protein molecular weight standard, 2-no IPTG induced whole bacteria component, 3-IPTG induced whole bacteria component, 4-supernatant component, 5-periplasmic cavity component, 6-cytoplasm soluble component, 7-inclusion body component;

FIG. 2B shows the SDS-PAGE and Western Blot results after purification and renaturation of EL-defensin fusion protein, 1-protein molecular weight standard, 2-purified EL-defensin protein, 3, 4, 5, 6-renaturation EL-defensin protein, 7, 8-EL-defensin fusion protein Western Blot results;

FIG. 2C shows the result of HLPC detecting the purity of the EL-defensin fusion protein.

FIG. 3 shows the results of an analysis of the binding capacity of the EL-defensin fusion protein in vitro. Wherein:

FIG. 3A shows the Western Blot results of EGFR expression levels in different tumor cells and J774A.1 cells;

FIG. 3B shows the result of ELISA immunoreaction analysis of EL-defensin fusion protein in A431 cells;

FIG. 3C shows the result of ELISA immunoreaction analysis of EL-defensin fusion protein in A549 cells;

FIG. 3D shows the result of ELISA immunoreaction analysis of EL-defensin fusion protein in H460 cells;

FIG. 3E shows the result of ELISA immunoreactivity analysis of EL-defensin fusion protein in J774.1 cells.

FIG. 4 shows the results of cellular immunofluorescence analysis of the binding activity of EL-defensin fusion protein to A431 cells. FIGS. 4A-4C show the effect of the fusion protein EL-defensin on A431 cells: 4A is cell nucleus DAPI coloring picture; 4B is a cell membrane coloring picture; 4C is a picture of the staining of the nuclei and membranes.

FIG. 5 shows the ability of the fusion protein EL-defensin to target A431 nude mice transplants tumors in vivo.

FIG. 6 shows a transmission electron microscope image of the morphological change of the fusion protein EL-defensin treated A431 cells. Wherein:

FIGS. 6A and 6B are photographs of A431 cells without any treatment;

FIGS. 6C and 6D are photographs of A431 cells treated with EL fusion protein;

FIGS. 6E and 6F are photographs of A431 cells treated with EL-defensin fusion protein.

FIG. 7 shows the results of comparison of the inhibition rates of the fusion proteins EL-defensin, LDP, EL and defensin on tumor cells.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Example 1Construction of recombinant expression vector pET 30-el-defensein

The recombinant plasmid pEL used in this example contains the EGFR oligopeptide ligand Ec gene and LDP gene, the construction method and its map are described in Kuo Xiao Fang, the construction of "oligopeptide targeting epidermal growth factor receptor and lidamycin-enhanced fusion protein and its anti-tumor activity", cancer, 2009, 28 (6): 561-.

5 primers were required for the construction of the recombinant vector pET 30-el-defensen:

E1（SEQ ID NO:8）：5’-gccat atgaaa tac ctg ctg ccg acc-3' (NdeI restriction sites underlined)

P2（SEQ ID NO:9）：5’-gcc gaa ggt cag agc cac gtg-3’

Pb1（SEQ ID NO:10）：5’-ggt gga ggc ggt tca ggt gga ggc ggt tca gat cattat aac tgc gtg tcc tcc ggc ggt cag tgt ctg tat agc gca-3’

Pb2（SEQ ID NO:11）：5’-gcctc gagctt gca aca ctt cgc ctt gcc acg ataaca ggt gcc ctg ggt ctt ggt aaa gat cgg gca tgc gct ata cag aca ctg-3' (XhoI restriction site underlined)

Pb4（SEQ ID NO:12）：5’-gcctc gagctt gca aca ctt cgc ctt-3' (XhoI restriction site underlined)

The recombinant plasmid pEL is taken as a template, and primers E1 and P2 are adopted to carry out conventional PCR reaction: pre-denaturation at 94 ℃ for 3 min, followed by denaturation at 94 ℃ for 30 sec, annealing at 56 ℃ for 30 sec, extension at 72 ℃ for 45 sec, 30 cycles of amplification reaction, and final extension at 72 ℃ for 10 min. The reaction system comprises:

the product was subjected to agarose gel electrophoresis, and a 501bp gene fragment carrying a signal peptide and designated ec-ldp (SEQ ID NO: 13) was recovered using the Omega gel recovery kit.

Pb1 is a5 'end primer, Pb2 is a 3' end primer, which are mutually templates, carrying out PCR amplification according to the following conditions to obtain a Human defensin β -1 (hBD-1) gene fragment (about 108 bp) (SEQ ID NO: 14) containing one enzyme cutting site, wherein the adopted PCR reaction conditions are that pre-denaturation is carried out at 94 ℃ for 3 minutes, then denaturation is carried out at 94 ℃ for 50 seconds, annealing is carried out at 55 ℃ for 50 seconds, extension is carried out at 72 ℃ for 50 seconds, 30-cycle amplification reaction is carried out, and finally extension is carried out at 72 ℃ for 8 minutes, and the reaction system comprises:

ec-ldp and hBD-1 are used as templates, primers E1 and Pb4 are used for carrying out PCR reaction, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, followed by denaturation at 94 ℃ for 50 sec, annealing at 65 ℃ for 50 sec, extension at 72 ℃ for 50 sec, 30 cycles of amplification reaction, and final extension at 72 ℃ for 10 min. The reaction system comprises:

the resulting gene fragment was named gene el-defensin (SEQ ID NO: 7).

The gene el-defensin comprises 3 parts except the connecting peptide, and an NdeI enzyme cutting site (6 bp), a coding sequence of Ec (66 bp) and a flexible peptide (GGGGS) are arranged from the 5 'end to the 3' end in sequence₂The coding sequence of (30 bp), the coding sequence of LDP (330 bp), flexible peptide (GGGGS)₂The coding sequence of (30 bp), the coding sequence of defensein (108 bp), and the XhoI enzyme cutting site (6 bp), wherein the total length is 576 bp.

NdeI and XhoI double enzyme digestion genes el-defensen and pET30a (+) vectors are respectively used for connection to obtain a recombinant expression vector which is named as pET 30-el-defensen and is transformed into escherichia coli DH5 α, positive clones are selected for sequencing, and the plasmid construction flow is shown in a figure 1.

In the resulting recombinant plasmid pET 30-el-defensen, an XhoI cleavage site was present at the 3' end of the defensin HBD-1 coding sequence, and pET30a (+) vector contained a histidine tag (6 amino acids, His-tag), so that the recombinant plasmid pET 30-el-defensen, when expressing the fusion protein, contained the His-tag to the protein for ease of purification and identification.

Example 2Expression and purification of fusion protein EL-defensin in Escherichia coli

Escherichia coli strain BL21star used in the present invention^TM(DE3) is a product of the company Novagen.

The recombinant expression vector pET 30-el-defensen constructed in example 1 was transformed into E.coli BL21, and a positive single clone was selected and inoculated into LB medium (kanamycin 50. mu.g/ml) and cultured overnight at 37 ℃. The sequencing is carried out, and a positive strain with correct sequencing and double enzyme cutting results is selected and stored at the temperature of-80 ℃ (the strain is Escherichia coli (which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: Beijing city rising district, Xingyang district, Xilu No. 1, institute of microbiology, China academy of sciences), 6 months and 4 days in 2013), and the strain preservation number is CGMCC No. 7676).

The positive monoclonal was inoculated into 5ml of LB medium, cultured overnight at 37 ℃ and when OD =1.0, 1mmol/LIPTG was added, induced for 8 hours, and the culture supernatant, the periplasmic cavity, the cytoplasmic soluble and insoluble fractions were analyzed according to the pET System operation manual (Novagen Co.). The molecular weight of the fusion protein EL-defensin is 19.8 KD. It was found that the EL-defensin protein was localized mainly in inclusion bodies (FIG. 2A) by 12% SDS-PAGE analysis, and the molecular weight size was consistent with that expected. Optimizing various conditions influencing the protein expression yield, and finally determining the expression conditions as follows: the temperature is 37 ℃, and the initial density of the thallus is A₆₀₀=1.0, IPTG concentration 1mmol/L, induction time 4 h.

The fusion protein is insoluble, renaturation is needed, the inclusion body protein component can be released after ultrasonic cracking treatment, and the EL-defensin fusion protein is provided with a His-tag label at the carbon end, so that a nickel column of His-tag5ml of GE company is adopted to carry out Ni2+ affinity chromatography purification according to the instruction of a kit. The protein was purified using different concentrations of imidazole, 5mM, 20mM, 50mM, and 250mM, respectively, and the protein eluted at 250mM imidazole was identified as the protein of interest EL-defensin (FIG. 2B) by 12% SDS-PAGE electrophoretic analysis, the molecular weight was consistent with the expected size.

The amino acid sequence of the obtained fusion protein EL-defensin is shown in SEQ ID NO. 5.

Example 3Renaturation of fusion protein EL-defensin

The soluble and purified EL-defensin protein was diluted to 15-50. mu.M with 6M urea in 1 Xbinding buffer, 2-mercaptoethanol was added to a final concentration of 10mM, and placed at room temperature for 30 min. The reduced protein was dialyzed overnight against at least 50 sample volumes of renaturation solution I (50mM Tris-HCl pH8.0,1mM EDTA,200mM NaCl,6M urea) to remove the reducing agent. And (3) carrying out fractional dialysis on the sample by using buffer solutions with urea concentrations which are sequentially decreased and have the same components as the dialysate I, wherein the urea concentrations are sequentially 3M, 2M, 1M, 0.5M and 0M. 750. mu.M oxidized glutathione (GSSG) and 400mM L-arginine were added at a urea concentration of 1M. Dialyzed at 4 ℃ overnight each time. And (4) dialyzing the sample obtained in the last step by using PBS with 50 times volume, and replacing the dialyzate once every 8-12h for three times. The dialyzed sample was filtered through a 0.45 μm filter and the filtrate was collected. The renatured sample was added to Ultrafree-MC membranes (Millipore, molecular weight cut-off: 3kDa) in an ultrafiltration centrifuge tube, and the protein was concentrated to an appropriate concentration by centrifugation at 3,500g and identified as the desired protein EL-defensin by 12% SDS-PAGE electrophoretic analysis (FIG. 2B), the molecular weight was consistent with the expected molecular weight. Purity of 85.4% was achieved by HPLC (FIG. 2C), and yield was approximately 30mg of non-renatured fusion protein per liter of fermentation broth.

Example 4Fusion protein EL-defensein vitro cell binding activity assay

Experiment (one):

human skin squamous carcinoma cell A431, human lung cancer cell A549, human lung cancer cell H460, human lung cancer cell PG-BE1 and mouse macrophage J774A.1 in logarithmic growth phase are washed with precooled PBS for 2 times, cells are digested with pancreatin, washed with PBS for 2 times, and then added with a proper amount of cell lysate (Beijing Baosi Biotech company) to BE cracked for 10 minutes on ice. After that, the mixture was centrifuged at 10,000rpm at 4 ℃ for 30 minutes, and the supernatant was collected.

The BCA kit is used for quantifying the fusion protein EL-defensin, 50 mu g of protein is mixed with a proper amount of 5 Xloading buffer solution, and the mixture is denatured in boiling water bath for 5 minutes and stored at-80 ℃ for later use.

Western Blot for detecting the expression level of EGFR of tumor cells shows that A431 is an EGFR high-expression cell strain, EGFR is expressed in A549 and PG-BE1, H460 expresses less EGFR, and J774A.1 does not express EGFR (figure 3A).

The immunogenicity of the fusion protein was examined by enzyme-linked immunosorbent assay (ELISA). A431, A549, H460 and J774A.1 cells were seeded at 1X 10 per well of 96-well plate⁴The cells were incubated at 37 ℃ for 24 hours, washed 3 times with PBS, and pre-cooled 0.05% glutaraldehyde (100. mu.l/well) at 4 ℃ was added to fix the cells at 4 ℃ for 30 minutes. The fixed cells were washed 3 times with PBS, blocked with 3% BSA solution at 4 ℃ overnight, and then washed 3 times with PBST buffer (0.05% Tween-20 in PBS). EL-defensein fusion protein (SEQ ID NO: 5), EL fusion protein (Ec and LDP fusion protein obtained by conventional method, SEQ ID NO: 15), LDP protein and HBD-1 protein (purchased from Abcam corporation) were diluted in multiple times into 96-well plates, each concentration was set to 3 parallel wells, and incubated at 37 ℃ for 2 hours. After washing 3 times with PBST, an anti-His-tag monoclonal antibody (diluted 1: 2500) was added and incubated at 37 ℃ for 2 hours. The plate was washed 3 times with PBST, and a horseradish peroxidase-labeled goat anti-mouse IgG antibody (1: 2500 dilution) was added and incubated at 37 ℃ for 2 hours. The plate was washed 5 times with PBST, and TMB substrate reaction solution (Beijing Tiangen Co.) (100. mu.l/well) was added thereto and reacted at room temperature for 10 to 30 minutes in the absence of light. The reaction was stopped with 100. mu.l of 2mol/L sulfuric acid per well and the absorbance at 450nm was immediately measured on a microplate reader. The results showed that the fusion proteins EL-defensin and EL reacted positively to a431, a549 and H460 cells expressing EGFR receptors (fig. 3B-D), j774a.1 cells did not have EGFR receptors on their surface, and EL-defensin reacted negatively (fig. 3E). While LDP and HBD-1 proteins without Ec ligand oligopeptide showed negative reaction in both EGFR expressing tumor cells and EGFR non-expressing tumor cells (FIGS. 3B-3E).

Experiment (ii):

the binding activity of the fusion protein EL-defensin and the A431 cells highly expressed by the EGFR is analyzed by a cell immunofluorescence method.

A431 tumor cells were cultured at 5X 10⁴The density of individual cells/well was inoculated into a six-well plate, cultured at 37 ℃ for 24 hours, washed 3 times with PBS, added with 70% methanol, fixed at 0 ℃ for 20 minutes, and washed 3 times with PBS. The fusion protein EL-defensin (100. mu.M/well) was added, incubated at room temperature for 2 hours or overnight at 4 ℃ and then washed 3 times with PBS. anti-His-tag antibody diluted at a ratio of 1:1000 was added thereto, and the mixture was reacted at room temperature for 2 hours, and washed 3 times with PBS. TRITC-labeled goat anti-mouse IgG (China fir gold bridge) diluted at a ratio of 1:50 was added, and the mixture was reacted at room temperature in the dark for 1 hour, and washed 3 times with PBS. 1:100 diluted DAPI (1 mg/ml) was added and the reaction was carried out for 15 minutes at room temperature with exclusion of light. PBS was washed 3 times and observed under a fluorescent microscope and photographed. As shown in the figure, red fluorescence was observed on the cell membrane of A431 cells, indicating that the EL-defensin fusion protein can bind to the cell membrane of tumor cells expressing EGFR receptor (figure)4）。

Example 5Small animal living body imaging analysis of in vivo targeting effect of fusion protein EL-defensin

The protein to be crosslinked (concentration 5 mg/mL) was dialyzed against the crosslinking reaction solution (borax buffer) at 4 ℃ for 3 times until pH 8.5. The preparation method of the crosslinking reaction liquid comprises the following steps: 19.08g Na₂B₄O₇10H₂O, 9.01g NaCl and HCl to adjust the pH value to 8.5, and water is added to the solution to reach a constant volume of 1 liter. DyLight680 (Thermo Scientific, DyLight680 catalog # 22859) was dissolved in DMF at a concentration of 10 mg/mL. DyLight680 used for each cross-linking should be freshly prepared and protected from light.

DyLight680 was added slowly to the protein solution as provided, gently shaken while adding to mix well with the protein, and reacted in a flat shaker dark at room temperature for 4 hours with gentle shaking. The cross-linked material was dialyzed in PBS for more than four times until the dialysate was clear.

The nude mice are inoculated with the ectopic transplantation tumor A431 through the vein, and when the tumor volume grows to about 200mm³At this time, FITC-labeled fusion protein was administered at a dose of 250. mu.g/mouse via tail vein injection. By observing different times, the fusion protein EL-defensin is found to have obvious targeting effect in A431 tumor cells, the maximum enrichment is achieved at the tumor site within 4 hours, and the fluorescence of the fusion protein at the tumor site is hardly detected after 12 hours, which shows that the drug is metabolized in vivo (FIG. 5).

Example 6Fusion protein EL-defensin in vitro lysis tumor cell electron microscope analysis result

Culturing cells and collecting the cells; washing with 0.1M PBS at pH7.2-7.4 for 2 times at room temperature for 5 min; absorbing PBS cleanly, adding 500ul of 2.5% glutaraldehyde at 4 ℃ or room temperature for 2 h; washing with 0.1M PBS (phosphate buffer solution) at pH7.2-7.4 for 3 times (5-10 min/time); sucking out PBS, adding appropriate amount of 4% gelatin, mixing, centrifuging at 3000rpm for 10 min; sucking out supernatant, standing for 10min, and cooling at room temperature; cooling and solidifying the gelatin in a refrigerator at 4 ℃ for 30 min; adding 100-100 ul of 2.5% glutaraldehyde, fixing for 30 min-1 h, 3 times of PBS and 5-10 min/time; the gelatin without sample was cut under a microscope, and the portion with sample was cut into 1mm³Small piece, 2.5% pentadiAldehyde preservation (the above method for pretreatment of cultured cells, if cells can be centrifuged into clumps, gelatin encapsulation may not be required); PBS3 times, 5-10 min/times, 1-2% osmic acid at 4 deg.C or room temperature, fixing for 2 h; washing with 0.1M PBS or water for 3 times, 5-10 min/time; 30% ethanol, 10min, 4 ℃; 50% ethanol, 10min, 4 deg.C; 70% ethanol, 10min at room temperature; 90% ethanol, 10min, room temperature; 95% ethanol, 10min, room temperature; 100% ethanol, 10min × 3, room temperature; PO10min × 3; infiltration; embedding; polymerizing; slicing, and observing with a microscope. As shown in fig. 6, the a431 cell control group had intact cell membrane, normal cell organelles and mitochondrial morphology in cytoplasm, but after EL-defensein and EL protein treatment, the a431 cell morphology changed, EL-defensein changed most significantly, cell membrane invaginated, vacuole generated in cytoplasm, tumor cell began to be lysed, mitochondrial structure changed, bilayer structure was hardly observed, some gaps between mitochondrial ridges became large, and vacuole generated in some mitochondria.

Example 7CCK-8 kit for detecting killing activity of EL-defensin fusion protein on tumor cells in vitro

Skin squamous carcinoma cell A431 and lung carcinoma cell H460 cells in a logarithmic growth phase are taken and inoculated in a 96-well plate at a density of 3000 cells per well, and after culturing for 24H at 37 ℃, fusion protein EL-defensin or EL or LDP or defensin (Abcam) with different concentrations are added, wherein each concentration is provided with 3 parallel wells. After 24h incubation, 20. mu.L of CCK-8 solution was added to each well and incubation continued for 1 h. Measurement of Absorbance at 450nm with microplate reader (A)₄₅₀). Experimental setup no-drug control group and no-cell blank group, cell viability was calculated and IC50 value was calculated according to the following formula:

cytostatic rate =100- (addition group a)₄₅₀Value-blank group A₄₅₀Value)/(control A₄₅₀Value-blank group A₄₅₀Value) × 100%.

The results show that compared with EL, LDP and defensein, the EL-defensein protein has the most obvious antiproliferative effect on A431 tumor cells, the 10 mu M EL-defensein protein has 67 percent of inhibition rate on squamous carcinoma A431 cells, the EL is 48 percent, and the LDP protein is only 22 percent (FIG. 7A); the inhibition rate of EL-defensin protein on H460 tumor cells was 65%, and the inhibition rate of EL protein was only 45% (FIG. 7B). Therefore, the anti-proliferation effect of the EL-defensin protein on the EGFR high-expression A431 cell line is almost the same as that of H460; the EL-defensin protein is higher than that of EL. Thus, it was shown that defensin HBD-1 enhances the in vitro anti-tumor activity of the fusion protein.

Example 8Growth inhibition effect of fusion protein EL-defensin on human squamous cell carcinoma A431 nude mouse transplanted tumor

Taking 5 female BALB/c nude mice with the weight of 18-22g, inoculating human squamous cell carcinoma A431 cells to axilla subcutaneous tissues of the nude mice, and inoculating 1 × 10 cells to each mouse⁷And (4) cells. When the tumor mass is long enough, it is cut into 2X 2mm in sterile physiological saline³The tumor mass is transplanted under the skin of the right axilla of the nude mouse by a trocar, and the incision is stuck by collodion cotton. The tumor mass is 100mm long³When the size is large, the nude mice are divided into groups according to the tumor size and the body weight, so that the average value of the tumor size of each group is 100mm³And the average body weight of each group is close to that of each group, and each group contains 6 nude mice. When the tumor volume is 100mm³In this case, the fusion proteins Ec-LDP-Hr (see Guo Xiao Fang, Clin Cancer Res2010;16: 2085-. On day 8 after the first administration, the same dose was administered once more. Tumor diameter and body weight were measured every 3 days during the experiment according to the formula V = ab²And/2 calculating the tumor volume (a: the tumor major diameter, b: the tumor minor diameter), drawing a tumor growth curve, and observing the weight change. On day 33 of the experiment, animals were sacrificed, and tumor weight was measured and tumor inhibition rate was calculated. The results are shown in Table 1.

TABLE 1 tumor growth inhibition of human squamous cell carcinoma A431 by the fusion protein EL-defensin

The experimental result of a nude mouse A431 animal shows that the fusion protein EL-defensin has the growth inhibition effect on the squamous cell carcinoma A431. As shown in Table 1, the tumor inhibition rate was 88.1% at 33 days when the dose of the fusion protein EL-defensin was 10 mg/kg. In contrast, the Ec-LDP-Hr fusion protein is only 74.2%, and the inhibition rate of the lidamycin prosthetic group protein LDP alone is 52.4% under the condition of 10mg/kg, which shows that the fusion protein EL-defensin improves the treatment effect on the squamous carcinoma A431 tumor (compared with the LDP group, p is less than 0.05).

Animal experiment results show that compared with the double-target fusion protein Ec-LDP-Hr constructed in the laboratory, the EL-defensin has better tumor inhibition effect, and the defensin component in the defensin fusion protein is presumed to fully play a role in killing tumor cell warheads, so that the immunotoxicity of the fusion protein is increased, and the growth of tumors is better inhibited.

Example 9Growth inhibition effect of fusion protein EL-defensein on human lung cancer H460 nude mouse transplanted tumor

The test method and the number of administrations were substantially the same as in example 8, except that when the tumor was human lung cancer cell H460, and the nude mice were sacrificed at day 27, the results are shown in Table 2.

TABLE 2 tumor growth inhibition of human Lung carcinoma H460 by fusion protein EL-defensein

The result of an animal experiment of a nude mouse H460 shows that the fusion protein EL-defensin has a growth inhibition effect on the lung cancer H460. As shown in Table 2, at 27 days, the tumor inhibition rate was 80.9% at a dose of 10mg/kg of the fusion protein EL-defensin. In contrast, the Ec-LDP-Hr fusion protein is only 59.6%, and the inhibition rate of the lidamycin prosthetic group protein LDP alone is 41.2% under the condition of 10mg/kg, which shows that the fusion protein EL-defensein improves the treatment effect on the lung cancer H460 tumor (compared with the LDP group and the Ec-LDP-Hr group, p is less than 0.05).

The animal experiment results show that compared with the double-target fusion protein Ec-LDP-Hr constructed in the laboratory, the EL-defensin has better tumor inhibition effect. The fusion protein Ec-LDP-Hr only plays a role in animal transplants that highly express EGFR and Her2 receptors. For example, the inhibition rate of the A431 highly expressing EGFR receptor and the Ec-LDP-Hr is 74.2%, but the inhibition rate in the tumor H460 of the low expressing EGFR receptor and the Her2 receptor is only 59.6%, while the EL-defensin has good inhibition effect on the tumors of both expressing EGFR receptor and not expressing EGFR receptor, which indicates that the defensin HBD-1 well plays a warhead role.

Moreover, the fusion protein EL-defensin has good curative effect on A431 transplanted tumors and has good effect on other tumors such as lung cancer H460, which shows that the fusion protein has universal inhibition effect on tumor cells in animal bodies.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Claims (2)

1. An application of an anti-tumor fusion protein in preparing an anti-tumor medicament is characterized in that the anti-tumor medicament is used for treating or preventing squamous cell carcinoma or lung cancer, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 5.

2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID NO. 7.

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